Conventional two headed myosin can be found in virtually all eukaryotic cells. In muscle it provides the force for muscle contraction, while it appears to be involved in cytokinesis membrane capping, and cell motility in non-muscle cells. Its hexameric structure, consisting of two copies each of a regulatory or phosphorylatable light chain (RMLC), an "essential" or alkali light chain (EMLC), and a heavy chain (MHC) has been conserved from lower eukaryotes through humans. Based on in vitro studies of smooth muscle and non-muscle myosin, the RMLC appears to play an important role in regulating myosin activity. The function of the EMLC is less well understood. The long term goal of our research is to understand the specific roles played by the MLCs in motile processes. Because Dictyostelium is amenable to study at the molecular genetic, cell biological and biochemical levels, it provides a particularly good system for the analysis of non-muscle myosin function. Specific goals for this proposal are: Construction of Dictyostelium cell lines exhibiting altered MLC expression. We will employ our cloned MLC cDNAs and genomic clones to disrupt normal MLC expression by gene targeting using homologous recombination, expression of antisense RNA, and over-expression of MLC polypeptide. Characterization of the effects resulting from altered MLC expression in vivo. We will analyze the MLC deficient cell lines for altered motility properties of non-muscle cells. Investigation of MLC functional domains. Initially we will focus on the phosphorylation site of the RMLC, and a highly conserved domain we have identified in the EMLC. These sites will be modified by in vitro mutagenesis and the modified genes reintroduced and expressed in Dictyostelium. The resulting cell lines will be analyzed to determine the effects the specific alterations in the MLCs have on motility. As time and resources permit we will also begin to localize MLC domains which might be involved in the interaction between the heavy and the light chains. "Mix and match" experiments employing skeletal muscle, smooth muscle and other non-muscle MLCs may also provide new information regarding domains critical for MLC function. Characterization of the in vitro biochemical properties of myosin carrying mutagenized MLCs. Because the biochemical properties of purified myosin are well characterized, analysis of the in vitro biochemical properties of myosin carrying mutagenized MLCs, and correlation of these properties with the phenotypes exhibited by cells expressing mutagenized MLCs should provide us with a direct correlation between the biochemical properties of myosin and in vivo function of myosin in non-muscle cells.